Martin W H, Creutz C E
Department of Pharmacology, University of Virginia School of Medicine, Charlottesville 22908.
J Neurochem. 1990 Feb;54(2):612-9. doi: 10.1111/j.1471-4159.1990.tb01915.x.
Chromobindin A is a large, multisubunit protein that binds to chromaffin granule membranes in a Ca2+- and ATP-regulated manner. Ca2+ stimulates binding to the membrane, whereas ATP, in the the absence of Ca2+, is required for release of the protein from the membrane. We now report that spectral and HPLC data indicate that nucleotides are associated with the native chromobindin A complex and that the protein can bind two molecules of [3H]ATP in vitro. Chromobindin A also appears to be a novel nucleotide triphosphatase. ATPase activity was detected in fractions containing chromobindin A isolated by affinity chromatography, gel filtration, or ion exchange chromatography. Kinetic studies indicated that the Vmax is 44 nmol of Pi/mg/min and the Km is 0.115 mM, whereas the nonhydrolyzable ATP analog 5'-adenylylimidodiphosphate acts as a competitive inhibitor of this reaction with a Ki of 0.08 mM. The activity was found to be sensitive to protease treatment or to preincubation at 65 degrees C and was inhibited by Ca2+ or low pH. The ATPase activity was not inhibited by N-ethylmaleimide, N,N'-dicyclohexylcarbodiimide, vanadate, oligomycin, or azide.
嗜铬粒蛋白A是一种大型多亚基蛋白，它以Ca2+和ATP调节的方式与嗜铬粒颗粒膜结合。Ca2+刺激其与膜结合，而在没有Ca2+的情况下，ATP是该蛋白从膜上释放所必需的。我们现在报告，光谱和高效液相色谱数据表明核苷酸与天然嗜铬粒蛋白A复合物相关，并且该蛋白在体外可结合两分子的[3H]ATP。嗜铬粒蛋白A似乎也是一种新型核苷酸三磷酸酶。在通过亲和色谱、凝胶过滤或离子交换色谱分离得到的含有嗜铬粒蛋白A的组分中检测到了ATP酶活性。动力学研究表明，Vmax为44 nmol Pi/mg/分钟，Km为0.115 mM，而不可水解的ATP类似物5'-腺苷酰亚胺二磷酸作为该反应的竞争性抑制剂，Ki为0.08 mM。发现该活性对蛋白酶处理或在65℃下预孵育敏感，并受到Ca2+或低pH的抑制。ATP酶活性不受N-乙基马来酰亚胺、N,N'-二环己基碳二亚胺、钒酸盐、寡霉素或叠氮化物的抑制。
